This invention relates to the field of molecular genetics, particularly the identification and detection of certain nucleotide sequences.
Familial clustering of common disorders independent of known risk factors indicates that genetic epidemiology studies may provide important leads to understanding the pathogenesis of many complex diseases and help identify individuals at increased risk. The multifactorial nature of many complex diseases suggests that numerous genes, each with multiple alleles having small to moderate effects, may account for the majority of genetic variation involved in defining risk of common chronic diseases on a population basis. Nucleotide variations that are found in at least one percent of the populations are called single nucleotide polymorphisms, or simply SNPs. SNPs occur in roughly one of every 500 bases. Consequently, some 200,000 SNPs lie within coding regions of genes. Much of the genetic variation between individual humans that contributes to differences in susceptibility to disease is believed to reflect SNP variations in DNA (Risch and Merikangas, 1996). Some SNPs cause or strongly contribute to specific diseases. For example, sickle cell anemia is caused solely by the change of an A to a T in the gene encoding the xcex2-chain of hemoglobin. There are many reports of positive association of SNPs with complex diseases such as hypertension (Brand et al., 1998), or end-stage renal disease (Yu et al., 1998).
SNPs may also prove to be useful in pharmacogenomics, a new approach to drug design, testing and utilization. Here, the premise is that depending on their genetic makeup, individuals respond differently to particular drugs. On another front, the use of SNPs as biallelic genetic markers offers the promise of rapid, highly automated genotyping.
In existing SNP assays, PCR primers flanking each SNP locus to be interrogated are chosen from publicly available genomic sequence information. In one format, the forward PCR primers are designed such that the nucleotide at the 3xe2x80x2-end of the primer complements the base adjacent to the SNP site. The regions containing the SNP polymorphism are then amplified by PCR and the resulting products are purified prior to a primer extension reaction. The extension reaction uses each PCR product as template and fluorescent dye-labeled dideoxynucleotide triphosphates (ca. 100-fold excess) to identify the base present at each SNP site, i.e., the SNP alleles. Each primer extension experiment requires pair(s) of dye-labeled ddNTPs (e.g., R110-ddTTP and ROX-ddCTP for an A-to-G nucleotide change). The labeled extension products representing the SNP alleles are separated by capillary electrophoresis and detected by laser-induced fluorescence.
Various aspects of existing methods limit their efficacy in analyzing SNPs. For example, some of the following commercially available reagents are used in current SNP assays. Rhodamine dye-labeled terminators are available in 16 dye/base combinations from E.I. DuPont de Nemours and Co. ET-labeled terminators (i.e., Energy-Transfer-labeled terminators) can be purchased from Applied Biosystems (BigDye terminator premix kit), or from Amersham Pharmacia Biotech (DYEnamic ET terminator premix kit). A disadvantage of using the ET-labeled terminators is that they offer no flexibility in the choice of dye/base combinations. Only one set of four ET-labeled terminators is available with a particular ET-label on each base. Moreover, both BigDye and DYEnamic ET terminators use FAM derivatives as donors that provide relatively low signal strengths and spectral purity. Finally, these ET-labeled terminators are not readily available other than as components of kits.
Other problems associated with current SNP assays are as follows: 1) With excitation at one wavelength, single dye-labeled terminators give lower signal intensities than ET-labeled terminators; 2) both single dye-labeled and ET-labeled terminator assays provide no discrimination between the fluorescence emission of the extended target and of the reagents; 3) assays using ET-labeled primers likewise provide no fluorescence emission discrimination between extended and unextended primer; 4) purification is required to remove the large excess of unincorporated labeled-ddNTPs (or labeled primers) to avoid masking of the extended target peak. For research studies, such a purification step can be tolerated, but it needs to be eliminated in high throughput assays.
A template-directed dye-terminator incorporation (TDI) assay, a homogeneous DNA diagnostic solution assay based on fluorescence resonance energy transfer (FRET), has recently been developed. In this assay, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5xe2x80x2-FAM-labeled primer in the presence of allelic acceptor dye-labeled dideoxy terminators (Chen and Kwok, 1997a,b). The FAM-labeled primer is extended one base by the acceptor-labeled terminator specific for the allele present on the template. The reaction mixture is then analyzed for changes in fluorescence intensities without separation. This method detects the intramolecular FRET against a background of intermolecular FRET. A related dye-labeled oligonucleotide ligation (DOL) assay in which a donor dye-labeled common probe is joined to an allele-specific, acceptor dye-labeled probe by DNA ligase has also been developed (Chen and Kwok, 1998, 1999).
There are certain limitations associated with the TDI assay. First, in some instances only an overall fluorescence emission is measured, thus, no multiplexing of SNPs can be performed. Second, the efficiency of FRET is sensitive to the distance between the donor and acceptor in ET primers (Ju et al., 1995; Hung et al. 1997). The primers used in the TDI assay carry the donor dye (FAM) at the 5xe2x80x2-end. Moreover, the primer lengths typically are more than 18 nucleotides long. Therefore, the ET-labeled SNP products are formed with donor-acceptor dye pairs separated by more than 18 bases. This long spacing results in poor ET efficiency and requires that the extended primer be dissociated from the template before detection can occur. Also, with FAM as a donor, the residual donor fluorescence emission is relatively high. Third, this assay involves either awkward calculations that create variant thresholds for different loci (Chen and Kwok, 1997a), or awkward real-time fluorescence detection for each cycle during the primer extension (Chen and Kwok, 1997b). These are not suitable for high throughput assays.
The present invention provides a variety of methods for analyzing target nucleic acids having a variant site. The invention also provides kits for performing such methods within research, clinical and laboratory settings. The methods generally involve conducting template-dependent primer extension reactions to form an energy transfer labeled extension product if a non-extendible nucleotide provided in the extension reaction mixture is complementary to the nucleotide at the variant site. The extension product includes a donor and acceptor fluorophore that together form a pair. One member of the pair is borne by the primer and the other member by the non-extendible nucleotide. By controlling various parameters such as the position of the fluorophore on the primer and the type of fluorophores utilized, certain methods of the invention can enhance signal strength and purity. Further, certain methods can be performed without the need to dissociate extension product from the target nucleic acid, or to separate other reaction components from the extension product, prior to detection.
More specifically, certain methods for analyzing variant sites in nucleic acids of interest involve hybridizing a primer bearing a first fluorophore to a segment of the target nucleic acid to form a labeled hybrid, wherein the 3xe2x80x2-end of the primer hybridizes to the target nucleic acid immediately adjacent to the variant site. Template-dependent extension of the primer is conducted in the presence of a polymerase and at least one non-extendible nucleotide bearing a second fluorophore, whereby a double-labeled extension product is formed if the non-extendible nucleotide is complementary to the nucleotide at the variant site and the first and second fluorophore borne by the extension product are brought into an energy transfer relationship. The first and second fluorophore borne by the primer and non-extendible nucleotide comprise a donor and an acceptor fluorophore. The presence or absence of the double-labeled extension product is then detected, the presence or absence of double-labeled extension product indicating the identity of the nucleotide at the variant site.
Because the label on the primer is located so that the fluorophores borne by the primer and the non-extendible nucleotide are brought into an energy transfer relationship during the extension reaction, the extension product can be detected while it is still hybridized to the target nucleic acid. Presence of extension product can be detected by an increase in the emission associated with the acceptor fluorophore or a decrease in emission from donor fluorophore. Other analytical methods utilize primers including modified nucleotides or nucleotide subsitutes to incorporate a fluorophore into a desired position in the primer to facilitate energy transfer once labeled non-extendible nucleotide is incorporated into the primer.
Other methods utilize particular combinations of dyes to increase signal strength while minimizing background signal from unreacted components of the extension reaction. For example, in some methods, one member of the fluorophore pair is a donor that has a high extinction coefficient and a low fluorescent quantum yield. Such a fluorophore can be paired with an acceptor fluorophore that does not exhibit strong fluorescence emissions when excited at the wavelength used to excite the donor fluorophore. Examples of such combinations include cyanine dyes as a donor (e.g., CYA, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5) and rhodamine dyes as acceptor (e.g., R110, R6G, TAMRA, ROX, FAM, JOE, ZOE, TET, HEX, NAN, Texas Red, and Rhodamine Red).
The invention further provides methods for analyzing mutiple variant sites at the same time. Certain multiplexing methods comprise conducting a plurality of template-dependent extension reactions with different primers, wherein different primers hybridize adjacent different variant sites on target nucleic acids. Each extension reaction comprises: (i) hybridizing one of the different primers to a segment of a target nucleic acid, wherein the primer bears a first fluorophore and an optional secondary label and the 3xe2x80x2-end of the primer hybridizes to a target nucleic acid immediately adjacent to a variant site, (ii) contacting the primer with a polymerase and at least one non-extendible nucleotide bearing a second fluorophore, whereby a multi-labeled extension product is formed if the non-extendible nucleotide is complementary to the nucleotide at the variant site and the first and second fluorophore borne by the extension product are brought into an energy transfer relationship. The different primers bear different first labels and/or mass labels and different non-extendible nucleotides optionally bear different second fluorophores so that different extension products corresponding to different variant sites bear different pairs of fluorophores and/or different secondary labels. Following extension product formation, the presence or absence of the different extension products is detected. The fluorophore pair and/or secondary label borne by the extension product serves as an indicator of the identity the nucleotides at the variant sites. Extension products generated from different variant sites can be encoded by attaching different secondary labels to different primers for different extension reactions and/or differentially labeling the non-extendible nucleotides.